Cell and Tissue Research
Cell Tiss. Res. 194, 279-285 (1978)
9 by Springer-Verlag 1978
Cytochalasin B-Induced Changes in Concanavalin A-Activated Lymphocytes Vladimir Viklick~, Petr Drfiber, Petr Sima*, and Alena Lengerovfi Institute of MolecularGenetics,CzechoslovakAcademyof Sciences,Prague, Czechoslovakia
Summary. Cytochalasin B (CB) administered simultaneously with a mitogenic dose of concanavalin A (ConA) interferes with the activation process. This interference involves structural alterations of cellular membrane which do not include a reduced Con A-binding capacity. This conclusion is supported by the observation of deformities in both nuclear and cytoplasmic membranes in Con A-activated lymphocytes subsequently treated with CB. The high incidence of membrane blebs and pseudomyelin bodies in the cytoplasm points to a general effect of CB on the structural organization of membrane which may secondarily interfere with some specific event such as generation or transfer of signals for activation or cytokinesis. Key words: 14C-thymidine incorporation- Autoradiography- Multinucleated blasts - Electron microscopy - Deformities of cellular membranes.
The function of microfilaments in the regulation of various membrane phenomena has been well established. As reported recently (Nicolson, 1976) these structures may also be involved in the control of the topography of plasma-membrane glycoproteins. Binding of a mitogen to its glycoprotein receptors tends to induce changes in the distribution of microfilaments (Edelman, 1976); when microfilaments are destroyed by CB within the first hours following the administration of a mitogen, the lymphocyte-activatingeffect of the latter is abolished (Medrano et al., 1974). In addition to its microfitament-mediated effect, CB may act also directly, i.e., by affecting the turnover of membrane phospholipids (Resch, 1976). Lymphoid cells contain relatively few microfilaments (Yahara and Edelman, 1975) which nevertheless seem to be involved in the activating effect of mitogens. Depending on the dose and timing of CB following mitogen administration (Resch et al., 1976, Greene et al., 1976) the activation processes may be interfered with more or less Send offprint requests to: Dr. V. Viklick~,,Instituteof MolecularGenetics,Bud~jovickfi1083, 14220 Praha 4, Czechoslovakia * Institute of Microbiology,CzechoslovakAcademyof Sciences,Prague, Czechoslovakia
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strongly. Cytokinesis is definitely more sensitive to CB t h a n the activation process (Janossy a n d Greaves, 1975). I n the present study we f o u n d that (1) the presence of C o n A in the m e d i u m is n o longer required for the second a n d further nuclear divisions of once stimulated lymphocytes, a n d (2) CB tends to affect the m e m b r a n e ultrastructure of cells that are already b e y o n d the CB-sensitive phase of their activation process.
Materials and Methods Cell Cultures
Spleencells were cultured in serum-free and thymocytesin calf serum-supplementedmedia as described elsewhere (Drfiber et al., 1977). The cells were obtained from 2-month-old female mice of the strain C57BL/10ScSnPh. Con A-Induced Activation
Con A-induced activation was evaluated radiosotopically and morphologically. Each well of a fiatbottomed microtest II tissue culture plate (Falcon) contained 106 cells in serum-free or supplemented media to which respectively0.5 and 5 Ixgof Con A/ml (Pharmacia, Uppsala) was added and the cultures were maintained for 68 h. To some of the 3 h- or 24 h-old cultures was added CB (Serva, Heidelberg)or cr methyl-D-mannoside (~MM, Sigma, St.Louis) to give final concentrations of 1, 2, 4, or 8 ~tgl/mland 0.1 M respectively. A utoradiography
Two h before the cultures were harvested, 2 I~Ciof 3H-thymidine(spec. activity 24 Ci/mM) was added to each well. With the use of a Shandon-Elliot cytocentrifuge, cell preparations were made, coated with Kodak stripping film and followinga 2-weekexposition, they were stained with methyl green pyronine. Similar preparations for light microscopy were stained according to May-Griinwald Giemsa; 100-300 cells were classified from each slide. Electron Microscopy
Cell suspensions were fixed with glutaraldehyde at 35~C, postfixed in osmium tetroxide and embedded in Vestopal IV. Ultrathin sections stained with uranyl acetate and lead citrate were examined with a Tesla BS 500 electron microscope.
Results CB a d m i n i s t e r e d s i m u l t a n e o u s l y with C o n A has a m u c h stronger effect t h a n when given 3 h thereafter; the difference is all or n o t h i n g with a CB dose of 4 llg/ml (complete i n h i b i t i o n o f activation versus zero effect). F o r this reason, this dose was used in all further experiments (Fig. 1). U n d e r n o r m a l conditions, in C o n A-activated lymphocyte cultures there is a low percentage of multinucleated cells which are, as a rule, n o t more than binuclear. W h e n CB is added 24 h following C o n A, cytokinesis is incomplete resulting in a higher incidence of cells which m a y have u p to 9 nuclei (Table 1, Figs. 2-5). This
Concanavalin A-Activated Lymphocytes and Cytochalasin B
cpm x 1() 3 20
Fig. 1. Effect of various concentrations of cytochalasin B (CB) on Con A-inducible lymphocyte activation added either at the start of the culture (1) or 3 h later (2)
a c a dose (jug/ml)
Table 1. Effect of ~-methyl-D-mannoside (~MM) on the occurrence of cytochalasin B (CB)-induced multinucleated concanavalin A (Con A)-activated spleen cells Culture conditions Con A (5 ~tg/ml)
CB (4 ~tg/ml)
ctMM (0.1 M)
+ + + +
~o of blasts"
~ of multinucleated cells a
360+ 28 16,264+ 442 7,921 + 1016 16,721+1351 8,903+ 803
1.6+ 0.6 80.6+ 2.8 71.0+ 4.3 82.3+3.5 75.6+ 4.5
0 2.6+ 0.6 23.3+ 3.0 3.3+0.5 25.6+ 1.5
CB and ~tMM were added 24 h after Con A a
Mean + S.D., cpm = counts per min
t e n d e n c y w a s o b s e r v e d e v e n a f t e r t h e a d d i t i o n , s i m u l t a n e o u s l y w i t h C B , o f a M M (a specific i n h i b i t o r o f C o n A b i n d i n g ) w h i c h b y itself d o e s n o t i n t e r f e r e w i t h t h e a c t i v a t i o n p r o c e s s ( T a b l e 1). T h e c y t o p l a s m o f t h e m u l t i n u c l e a t e d cells is s t r o n g l y b a s o p h i l i c . Besides n o r m a l n u c l e i t y p i c a l f o r b l a s t cells, m i c r o n u c l e i a n d e v e n n u c l e a r p o w d e r w e r e o b s e r v e d (Figs. 3, 5). M i c r o n u c l e i w e r e o c c a s i o n a l l y c o n n e c t e d w i t h s o m e o f t h e n o r m a l nuclei by a fine b r i d g e (Fig. 3). T h e i n c i d e n c e o f o d d n u m b e r s o f nuclei i n d i c a t e s a s y n c h r o n y o f n u c l e a r d i v i s i o n s w h i c h is f u r t h e r
V. Viklick~ et al.
Figs. 2--9. Various examples of effects of cytochalasin B added 24 h after Con A to thymocyte cultures. Fig. 2. Blast with 9 nuclei and cytoplasmic bleb (process) x 1000. Fig.3. Blast with 3 nuclei, one connected with "micronucleus" by fine bridge (arrow) x 1000. Fig. 4. Blast with 4 asynchronous nuclei x 1000. Fig. 5. Trinucleated blast with heavily vacuolized cytoplasm x 1000. Fig. 6. Blebbing of nuclear envelope, N nucleus; CYcytoplasm x 27,000. Fig. 7. Blebs of plasma membrane (arrow) x 45,000. Fig. 8. Pseudomyelin bodies close to plasma membrane (arrow) x 36,000. Fig. 9. Pseudomyelin figures (arrow) and multivesicular bodies (arrowhead) in vicinity of nucleus (N) x 36,000
reflected in the fact that individual nuclei within a cell appeared in various phases of the mitotic cycle as shown both morphologically (Fig. 4) and autoradiographically. The effect of CB on the Con A-inducible activation process is, however, not through its affecting the capacity of the plasma membrane to bind Con A (unpublished results, Greene et al., 1976).
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Ultrastructurally, among the Con A-stimulated and CB treated cells many deformities of the nuclear and cytoplasmic membranes were seen. The former displayed either separation of the two layers of the nuclear envelope or blebs with central cytoplasm and a small area of the nucleus surrounded by a membrane (Fig. 6). Multiple blebs associated with plasma membrane (Fig. 7) in some cases formed tunnel-like structures. The cytoplasm of the multinucleated cells contained many free ribosomes and occasionally granular endoplasmic reticulum as is typical for T-blast cells. In many cells membranous structures resembling myelin figures, frequently with a homogenous electron-dense center, were present. Such pseudomyelin bodies occurred both close to the plasma membrane (Fig. 8) and to the nucleus (Fig. 9). In Con A-stimulated cells (regardless of being CB-treated or not), abundant multivesicular bodies were observed particularly in the Golgi region. Discussion
Binding of Con A to plasma membrane receptors of lymphocytes in vitro induces various topographic, physical and biochemical changes in the membrane; the "new" membrane configuration seems to mediate the mitogenic effect of Con A on the T cell subpopulation. If the membrane remains "frozen" in this state, as seems to be the case with transformed cells, the signal for DNA synthesis tends to persist (Burger, 1973, Smets, 1973). In our present experiments, the asynchronous nuclear divisions in multinucleated cells indicate, however, that (1) the responsiveness, i.e. DNA synthesis of individual nuclei within the same cell may differ and (2) the presence of Con A on the plasma membrane is no longer needed for second and further nuclear divisions within a given cell (Table 1). The complex effect of CB on the cell includes depolymerization of microfilaments and a number of other changes (for review see Copeland, 1974). When given in adequate dose at the same time as Con A, CB interferes with its activating effect without preventing Con A binding to its membrane receptors. During the first hours of lymphocyte activation, the plasma membrane was found to undergo a number of physicochemical and biochemical changes (for review see Resch, 1976); their interrelationship and relative significance for activation to take place remain, however, to be elucidated. Con A-stimulated lymphocytes (as compared to resting cells) offer more favourable conditions for the various CB inducible changes to be detectable directly in the target organelles. The most distinct change we observed in the cytoplasm of multinucleated cells was the presence of pseudomyelin structures which tend to accompany membrane disorganization in a number of situations (for review see Avcyn and Schachlamov, 1975). Another distinct feature is the abundance of blebs of both plasma and nuclear membranes. The latter type was repeatedly reported for thymocytes and stimulated T cells (T6r6 and O1/th, 1966, Sebuwufu, 1966; Biberfeld, 1971), and thought to reflect functional differentiation of the cells. One wonders whether formation of these blebs (as well as of pseudomyelin bodies) may be generally associated with the production of cytomembranes; their increased incidence in CB-induced multinuc-
Concanavalin A-Activated Lymphocytes and Cytochalasin B
leated cells would then simply reflect a defect in the structural integration of newly synthesized membranes. Blebs and similar configurations of cellular membrane were described among cytochalasin D effects in other experimental models (Godman et al., 1975). We found no multivesicular blebs as frequently observed in PHA-stimulated lymphocytes (Biberfeld, 1971a, b; Janossy et al., 1977), but multivesicular bodies (which are thought to result from endocytosis of ligandreceptor complexes) were fairly frequent. Nevertheless, the increased incidence of such structures induced by CB in proliferating lymphocytes points to a general effect of the drug on the structural organization of membranes; the induced general changes may then interfere with some specific processes including generation or transfer of signals for various steps in lymphocyte activation.
References Avcyn, A.P., Schachlamov, V.A.: Changes in ultrastructure and permeability of cell membranes in pathology. Biophysik 5, 204-243 (1975) Biberfeld, P.: Endocytosis and lysosome formation in blood lymphocytes transformed by phytohaemagglutinin. J. Ultrastruct. Res. 37, 41-68 (1971a) Biberfeld, P.: Morphogenesis in blood lymphocytes stimulated with phytohaemagglutinin (PHA). Acta path. microbiol, scand. A 223, 1-70 (1971 b) Burger, M.M.: Surface changes in transformed cells detected by lectins. Fed. Proc. 32, 91-100 (1973) Copeland, M.: The cellular response to cytochalasin B: a critical overview. Cytologia 39, 709-727 (1974) Drfiber, P., Viklick~, V., Lengerov~i, A.: Steps in mitogenic action of concanavalin A. I. Colchicine- and cytochalasin B-sensitive events. Fol. biol. (Praha) 23, 305-316 (1977) Edelman, G.M. : Surface modulation in cell recognition and cell growth. Science 192, 218-226 (1976) Godman, G.C., Miranda, A.F., Deitch, A.D., Tanenbaum, S.W. : Action of cytochalasin D on cells of established lines. III. Meiosis and movements at the cell surface. J. Cell Biol. 64, 644-667 (1975) Greene, W.C., Parker, Ch.M., Parker, Ch.W.: Cytochalasin sensitive structures and lymphocyte activation. Exp. Cell Res. 103, 109-117 (1976) Janossy, G., Greaves, M.: Functional analysis ofmurine and human B lymphocyte subsets. Transplant. Rev. 24, 177-236 (1975) Janossy, G., Shohat, M., Greaves, M.F., Dourmashkin, R.R.: Lymphocyte activation. IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro. Immunology 24, 211-227 (1972) Medrano, E., Piras, R., Mordoh, J.: Effect of colchicine, vinblastine and cytochalasin B on human lymphocyte transformation by phytohemagglutinin. Exp. Cell Res. 86, 295-300 (1974) Nicolson, G.L.: Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over cell surface components. Biochim. biophys. Acta (Amst.) 457, 57-107 (1976) Resch, K.: Membrane associated events in lymphocyte activation. In: Receptors and recognition, Ser. A, Vol. 1 (P. Cuatrecasas and M.F. Greaves eds.), pp. 61-117. London: Chapman and Hall 1976 Resch, K., Prester, M., Ferber, E., Gelfand, E.W.: The inhibition of initial steps of lymphocyte transformation by cytochalasin B. J. Immunol. 117, 1705-1710 (1976) Sebuwufu, P.H.: Nuclear blebs in the human foetal thymus. Nature (Lond.) 212, 1381-1383 (1966) Smets, L.A.: Membrane of transformed cells is not permanently in mitotic configuration. Nature (Lond.) New Biol. 245, 113-114 (1973) T6r6, I., Olfih, I.: Nuclear blebs in the cells of the guinea-pig thymus. Nature (Lond.) 212, 315-317 (1966) Yahara, I., Edelman, G.M.: Electron microscopic analysis of the modulation of lymphocyte receptor mobility. Exp. Cell Res. 91, 125-142 (1975)
Accepted July 3, 1978